Fig. 3

ZDHHC18 and ZDHHC23 target different subsets of glioma stem cells (GSCs) in specific niches. a, b Immunofluorescence images showing ZDHHC18 (red) and ZDHHC23 (green) positive locations and cells in the leading edge (a) and neurotic (b) regions of the glioblastoma (GBM) samples. Scale bar (upper image): 200 μm; (lower image, inset box): 50 μm (white). Quantification of ZDHHC23 positive or ZDHHC18 and ZDHHC23 positive cells in three fields is presented. Error bars represent the SEM. c Results of flow cytometry analysis showing the relationship between the expression of ZDHHC18 (or ZDHHC23) and stem cell marker CD133 (or CD44) in the leading edge and neurotic regions of the GBM samples. Quantification of ZDHHC18/CD133, ZDHHC23/CD133, ZDHHC18/CD44, and ZDHHC23/CD44 positive cells in three fields is presented. Error bars represents the SEM. d Expression levels of ZDHHC18 and ZDHHC23 detected by western blot analysis in neural progenitor cells (NPC1 and NPC2), proneural GSCs (PN12, PN16, and PN19), and mesenchymal GSCs (MES23, MES27, and MES29). β-actin was used as a loading control. e Bar graph showing Pearson coefficients of correlation between ZDHHC18 (blue) or ZDHHC23 (red) mRNA expression and the mature vasculature, microvasculature, and hypoxia activation signatures in 12 GSC and one NPC culture (**, p < 0.01; ***, p < 0.001). f Cell viability of NPC1, proneural GSCs (PN12), mesenchymal GSCs (MES23), or proneural (or mesenchymal) GSCs transfected with indicated plasmids was determined under baseline, low-glucose, hypoxia, or conditions with a combination of these stresses. Data are presented as means ± SEM (*, p < 0.05; **, p < 0.01; ***, p < 0.001). g and h Heatmap showing the molecular subtype marker expression in proneural GSCs (PN12, PN16, and PN19), mesenchymal GSCs (MES23, MES27, and MES29), and proneural (or mesenchymal) GSCs transfected with indicated plasmids under baseline or low-glucose/hypoxia stress. Proneural markers: DLL3, OLIG2, ASCL1, CD133, and SOX2; mesenchymal markers: CD44, CHI3L1, TIMP1, and TGFβ1. Z-scores were calculated from the ΔCt values obtained in the qPCR analysis